During development and differentiation, genes become competent to be expressed or are stably silenced in an epigenetically heritable manner. This selective activation/repression of genes leads to the differentiation of tissue types. Recent evidence suggests that modifications of histones in chromatin contributes substantially to determining whether a gene will or will not be expressed. Our group is interested in understanding how chromatin-modifying protein complexes get recruited to the DNA. In Drosophila, two groups of genes, the Polycomb group (PcG) and the Trithorax group (TrxG) are important for inheritance of the silenced and the active chromatin state, respectively. Regulatory elements called Polycomb group response elements (PREs) are cis-acting sequences required for the recruitment of chromatin-modifying PcG protein complexes. Recently it has become clear that TrxG proteins act through either the same or overlapping cis-acting sequences. Our group is working on understanding how PcG and TrxG proteins get recruited to the DNA. [unreadable] [unreadable] Polycomb group response elements (PREs) are DNA elements through which the Polycomb group (PcG) of transcriptional repressors act. Many of the PcG proteins are associated in two protein complexes that repress gene expression by modifying chromatin. Both of these protein complexes specifically associate with PREs in vivo, however, it is not known how they are recruited or held at the PRE. PREs are complex elements, made up of binding sites for many proteins. Our laboratory has been working to define all the sequences and DNA binding proteins required for the activity of a 181-bp PRE from the Drosophila engrailed gene. At least 9 binding sites are present within this 181-bp PRE. Two of the binding sites are for the Polycomb-group proteins Pleiohomeotic (Pho) and Pleiohomeotic-like (Phol). The proteins GAGA factor and Pipsqueak bind to another two sites. The DNA binding proteins Zeste and Dsp1 also are present within the engrailed PRE we study. Proteins that bind to the other three sites were unidentified. We have recently found that one of the sites necessary for PRE activity, Site 2, can be bound by members of the Sp1/KLF family of zinc-finger proteins. This family of proteins encodes transcription factors and has been extensively studied in mammals. There are 20 Sp1/KLF family members in mammals. In Drosophila there are 10 Sp1/KLF family members, and nine of them bind to Site 2. We derived a consensus-binding site for the Sp1/KLF Drosophila family members and show that this consensus sequence is present in most of the molecularly characterized PREs. These data suggest that one or more Sp1/KLF family members play a role in PRE function in Drosophila. During the past year we have been working on determining which of the Sp1/KLF family members in Drosophila may be involved in PcG function. We are in the process of making antibodies to the three most likely candidates. We will then determine the expression patterns of the proteins in embryos, and do chromatin immunoprecipitation experiments to determine whether they are bound to PREs. The genes for factors bound to PREs will be mutated and the phenotypes of the mutants analyzed for possible PcG phenotypes.[unreadable] [unreadable] The Drosophila engrailed gene encodes a homeodomain protein that plays an important role in the development of many different parts of the embryo including the formation of the segments, nervous system, head, and gut. It also plays a very important role in the development of the adult, specifying the posterior compartment of each imaginal disk. Accordingly, engrailed is expressed in a very specific and complex manner in the developing organism. The 181-bp engrailed PRE we have been studying is located near the engrailed promoter from ?576 to ?395 upstream of the transcription start site. We are interested in determining the role of this PRE in the control of engrailed expression. One of the first things we learned in our studies is that this PRE is redundant with other flanking PREs in the endogenous engrailed gene. There is another strong PRE located from ?1100 to ?1500 and probably other weak PREs nearby. In fact, when we examined the location of Ph and Pho proteins on engrailed DNA by chromatin immunoprecipation (ChIP), we found that they are bound to a 2.5 kb region extending from the engrailed promoter to about ?2.5kb upstream. Therefore, it is perhaps not too surprising that a 500bp deletion that includes the 181-bp PRE and flanking sequence did not lead to ectopic engrailed expression. The remaining PREs were apparently sufficient to recruit PcG proteins. However, what was surprising to us was that loss of this DNA lead to a loss of function phenotype, suggesting that this DNA must also play a positive role in the expression of engrailed. Recent experiments suggest that there are multiple positive elements either overlapping with or coincident with the PREs. We are working on determining whether and positive and negative sequences can be separated. The regulatory sequences for the engrailed gene extend over a 70 kb region. Our lab has used reporter constructs to find sequences important for expression in stripes, the nervous system, the head, etc. There are discrete regulatory elements located throughout the 70kb region. We also find at least 7 additional PREs located throughout the region. PcG protein complexes have been shown in vitro to bring together DNA fragments, and it is possible that they cause looping in vivo. We are interested in learning whether the additional PREs are involved in mediating interactions between distant enhancers and the engrailed promoter.